Nucleic acids are measured to identify molecules of a specific target nucleic acid sequence in a population of heterogeneous nucleic acids, DNA or RNA, or to measure products of reactions where nucleic acids, DNA or RNA, are modified. Such measurements are generally permutations of the following procedures:
a. where the starting nucleic acid is RNA, conversion to DNA is accomplished by a reverse transcription reaction. The oligonucleotide primers for the reverse transcription reaction may be specific for the target sequence or may be general for conversion of all RNA sequences to DNA;
b. amplification of the target nucleic acid by target sequence specific reactions. These include polymerase chain reaction (PCR) with sequence specific primers, and primer extension reactions again with a target sequence specific oligonucleotide primer. Rolling circle amplification of DNA has also been used to amplify specific DNA sequences;
c. physical separation of the heterogeneous nucleic acids. Such physical separations include but are not limited to size fractionation and affinity separation when amplified nucleic acids are produced with derivatized substrates including but not limited to biotinylated deoxyribonucleotide triphosphates;
d. labeling of the nucleic acid. As mentioned in c. above, amplified nucleic acids may be labeled using either derivatized deoxyribonucleotide triphosphates or derivatized oligonucleotide (RNA or DNA) primers; and
e. detection of the nucleic acids. Nucleic acids can be detected either through the labeling moiety, or by physical separation followed by detection with nucleic acid specific dyes.
One of the more common methods for the quantitative detection of target sequences is the sequence specific amplification of the target sequence(s) by PCR, either from DNA or from cDNA after reverse transcription, physical separation by gel or capillary electrophoresis, and detection by fluorescent labeling (e.g. of dsDNA by ethidium bromide or by use of fluorescently labeled primers in the amplification). Another common technique for the quantitative detection of target sequence(s) involves “real time” PCR.
PCR technology is widely used to aid in quantitating DNA because the amplification of the target sequence allows for greater sensitivity of detection than could otherwise be achieved. The point at which the fluorescent signal is measured in order to calculate the initial template quantity can either be at the end of the reaction (endpoint QPCR) or while the amplification is still progressing (real-time QPCR). The more sensitive and reproducible method of real-time QPCR measures the fluorescence at each cycle as the amplification progresses.
The reporter molecule used in real-time QPCR reactions can be (1) a sequence-specific probe composed of an oligonucleotide labeled with a fluorescent dye plus a quencher or (2) a non specific DNA binding dye that fluoresces when bound to double stranded DNA.
Both of these techniques, and others not described in detail, require instrumentation either for physical separation or detection. The requirement for instrumentation and/or separation technologies with their attendant sample handling limits the use of quantitative and qualitative target sequence detection. Accordingly, there is a need for methods of detecting and measuring nucleic acids that do not require expensive, delicate instrumentation either for sample separation or for detection. Such measurements include but are not limited to the identification of molecules of a specific nucleic acid sequence as well as the detection of nucleic acids that are the product of nucleic acid modifying reactions. Nucleic acid modifying reactions include but are not limited to polymerization reactions, ligation reactions, nuclease reactions and recombination reactions.
Fluorescent Intercalating Nucleic Acid Dyes
A common method for the detection of nucleic acids is by staining them with fluorescing intercalating dyes. These dyes have several unique features that make them especially useful: 1) They have a high molar absorptivity; 2) Very low intrinsic fluorescence: 3) Large fluorescent enhancements upon binding to nucleic acids; and 4) Moderate to high affinity for nucleic acids, with little or no staining to other biopolymers. Intercalating nucleic acid stains have fluorescence excitations and emissions that span the visible-light spectrum from blue to near-infrared with additional absorption peaks in the UV, making them compatible with many different types of instrumentation. These dyes are excited with an extrinsic light source that has a spectrum that overlaps with the maximally excitation wavelength of the intercalated dye. They may be used to image both RNA and DNA. Some commonly used dyes are listed below.
Dye NameEx/Em*ApplicationEthidium Bromide300/600Quantitation and Detection of dsDNAEthidium Bromide510/620Quantitation and Detection of dsDNAHomodimer-1PICOGREEN ®502/523dsDNAQuantitation ReagentOLIGREEN ®498/518Quantitation and Detection of ssDNA andQuantitation ReagentoligonucleotidesRIBOGREEN ®500/520Quantitation and Detection of RNAQuantitation ReagentSYBR GOLD ® stain495/537Quantitation and Detection of single- ordouble-stranded DNA or RNA post-electrophoresisSYBR GREEN I ® stain494/521Quantitation and Detection of double-strandedDNA and oligonucleotides post-electrophoresisAlso useful for real-time PCR assaysSYBR GREEN ® stain492/513Sensitive stain for RNA and single-strandedDNA post-electrophoresisSYBR SAFE ® stain502/530Sensitive DNA gel stain with significantlyreduced mutagenicitySYBR DX DNA BLOT ®475/499Sensitive stain for DNAstain*Excitation (Ex) and emission (Em) maxima are the wavelength, in nanometers, (nm) of light that maximally excites the intercalated dye and the wavelength of light that is maximally emitted when the dye fluoresces, respectively.Resonance Energy Transfer
Energy may be donated to nucleic acid intercalated dye either by photons or by resonance energy transfer. The principle of energy transfer between two molecules can be exploited as a means to provide information about relative changes in their proximity and orientation to one another. Resonance Energy Transfer (RET) is the transfer of excited state energy from a donor to an acceptor molecule. Förster resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. This can only occur if the absorption spectrum of acceptor molecule overlaps with the emission spectrum of the donor. Förster determined that the degree of resonance energy transfer between the energy donor and energy acceptor is inversely proportional to the distance between the two molecules to the sixth power. In the case of FRET, an external light source of specific wavelength is used to excite the donor molecule.
Bioluminescent Resonance Energy Transfer (BRET) uses biological molecules such as a luciferase as the donor molecule. Depending on the species of origin, luciferases that use coelenterazine as a substrate generate blue light in the range of 450 to 500 nm. When a suitable acceptor is in close proximity, the blue light energy is captured by RET. The acceptor molecules are generally a class of proteins that have evolved the ability to be excited by blue light and then fluoresce in longer wavelengths typically with maximal spectral emissions above 500 nm. In both FRET and BRET the molecules of interest may be either covalently or non-covalently linked or brought in to proximity by conformational change or by spatial migration or by an alteration in their relative orientations to one another. For instance, the two molecules may be conjugated to two separate proteins of interest. They may then be brought into proximity by their affinity for one another or their affinity for a third molecule. They may also be attached to a protein of interest and then brought closer due to a conformational change within the protein of interest. Generally the two molecules must be within 100 Å of one another for resonance energy transfer to occur and changes as little as 1-2 Å may be detected. Luciferases that have been used in BRET include those from the firefly, Renilla reniformis and Gaussia princeps. A commonly used fluorescent protein is the green fluorescent protein (GFP) from Aequorea victoria. BRET is generally used to measure the degree of affinity or degree of conformational change between two protein domains either covalently or non-covalently linked.